The role of mitochondrial fusion is disputed in cardiac myocytes;some believe fusion has no role in these cells where mitochondrial contact is enforced by physical constraints, some believe it is strictly pathological because it promotes SR-mitochondrial calcium transport that opens mitochondrial permeability transition pores, and some contest that mitochondrial fusion is essential to mitochondrial health. There are currently no published in vivo data addressing this issue and unpublished results using mouse knockouts are inconsistent. In this revised R21 we propose studies that will use in vivo genetic manipulation of mitochondrial outer and inner membrane fusion proteins to define the role of mitochondrial fusion in Drosophila heart tubes. We will use a combination of cardiac gene manipulation, cardiac phenotyping, and live cardiomyocyte research using new Drosophila melanogaster (fruit fly) cardiomyopathy models and fluorescent protein indicator models that we have generated. We will use genetic complementation, substituting human mitofusins for suppressed Drosophila mitochondrial fusion protein, to interrogate the roles of mammalian mitochondrial fusion proteins. The Drosophila heart tube is (at the cellular, transcriptional, and functional level) sufficiently like the mammalian heart to permit analysis of cardiac structure and function using analogous phenotyping techniques, permitting extrapolation of such complementation/rescue data to higher organisms. Because viable Drosophila heart tubes can be maintained for many hours at room temperature and normal atmosphere, they are optimal for live cardiomyocyte studies. This combination of mechanistic cell biology and intact cardiac physiology is not possible in mammalian systems. The proposed studies will not address an important unanswered scientific question, but will generate new reagents and techniques for the scientific community that will broadly advance cardiac studies using the Drosophila platform. Indeed, the current studies are an ambitious test of the capabilities of hypothesis testing research in the Drosophila cardiac system. Our Specific Aims are: SA#1. Generate novel Drosophila genetic models with cardiac-specific mitochondrial fusion defects analogous to the cardiac-specific mitofusin null mouse models we and others are developing. SA#2. Develop and deploy innovative techniques for analysis of resulting cardiac phenotypes in ways not possible with mouse models through the use of novel fly lines expressing genetically-encoded fluorescent probes for cardiomyocyte calcium signaling, mitochondrial and sarcoplasmic reticular morphometry and interactions, ATP/ADP ratio, ROS (hydrogen peroxide) production, and mitochondrial redox potential. PUBLIC HEALTH RELEVANCE: These studies will begin to answer important fundamental questions about the role of mitochondrial fusion and mitochondrial-sarcoplasmic tethering on in vivo cardiac function. The models, techniques and approaches described herein will be applicable to any future genetic cardiomyocyte-specific manipulations in Drosophila melanogaster, and will establish a useful new high-throughput analytical platform for studies requiring cardiac- specific gene manipulation and phenotyping.